Biological research has benefited enormously from the study of genes isolated by cDNA cloning techniques. Since their introduction, (1,2) these techniques have been refined to allow for the isolation of eukaryotic genes expressed at low frequencies. Screening of bacterial libraries using oligonucleotide probes or antibodies to eukaryotic gene products are widely used techniques (3-7). Despite advances, the construction of cDNA libraries still remains an arduous task, particularly if one wishes to construct a directional cDNA library. In directional cloning the vector and fragments to be cloned share an oriented pair of unique endonuclease restriction sites at their termini, which offers efficient cDNA capture and predictable insert orientation. Methods for constructing directional libraries in plasmid vectors exist (3,4,5). However, if phage vectors, particularly bacteriophage vectors, could be used for constructing directional libraries they would greatly simplify the task of constructing and screening large cDNA libraries (6,7). We have developed a new phage vector for the construction of directional libraries that retains many of the useful features of existing bacterial expression vectors.
A directional library constructed in phage would offer several advantages relative to the non-directional libraries constructed with existing phage vectors. The directional library would contain a small number of non-recombinant phage and few, if any, phage would contain cDNA fragments inserted in the incorrect orientation, the directional library would be more efficient in the capture of cDNA fragments due to the high vector:insert ratio, after cleaving the directional vector with endonucleases at the cloning site, subsequent treatment with phosphatase to insure high insertion rates is unnecessary, and DNA inserts in the vector can be cloned in a known orientation relative to vector encoded expression signals.